Replicated chromosomes (sister chromatids) acquire structural features that are essential for their segregation during mitosis. Each sister chromatid has a centromere, a protein DNA complex that mediates attachment and movement of chromosomes on the microtubules of the mitotic spindle. In addition, sister chromatids are paired; this pairing is part of the mechanism that ensures the direction of movement of sister chromatids and regulates the timing of segregation. Finally, sister chromatids are condensed; this shortening may allow chromosomes to move without becoming entangled and prevents chromosome ends from crossing the plane of cytokinesis at the end of mitosis. To elucidate the molecular basis of these three features of sister chromatids, we are using the yeast, Saccharomyces cerevisiae, as a model system. We have already developed an in vitro assay for centromere binding to microtubules and an in vivo assay for monitoring sister chromatid pairing and condensation. We have also used mutants to identify genes that encode potential structural or regulatory components of centromeres, sister chromatid pairing and chromosome condensation. Here, we propose to exploit our assays and these components to understand the assembly of the centromere, the mechanism of its microtubule-associated activities, and the role of the centromere in sister chromatid pairing. We will also use mutations that cause precocious disassociation of sister chromatids as a means to identify and characterize structural or regulatory components of sister chromatid cohesion. Finally, we will study two members of the newly-discovered SMC family, a conserved set of chromosomal proteins that modulate higher order chromosome packaging from bacteria to man. We will attempt to reveal the biochemical activities of SMC proteins as well as to identify and characterize SMC-associated proteins in vivo. Our findings will not only help understand mitosis but will also provide insights into other microtubule-mediated processes, the regulation of the cell cycle and the role of chromosome structure in gene expression. Furthermore, since mutations that cause aberrant chromosome transmission also enhance the development of cancers, some cancers will probably involve mutations that perturb mitotic chromosome structure. Moreover, at least one protein involved in chromosome condensation is already established as a successful target for chemotherapy. Therefore, our studies of mitotic chromosome structure are likely to have both broad implications in basic research and applied applications in biomedicine.